鲍曼不动杆菌CRP蛋白通过调控cas3基因表达增强细菌毒力的机制

cAMP receptor protein enhances Acinetobacter baumannii virulence by regulating cas3 expression

摘要:

目的 探究鲍曼不动杆菌转录调控因子cAMP受体结合蛋白(cAMP receptor protein, CRP)对cas3基因的功能调控作用。方法 利用原核表达系统表达并纯化CRP蛋白，采用电泳迁移率变动分析(electrophoretic mobility shift assay, EMSA)实验探究CRP对cas3启动子的结合作用，并通过qPCR明确CRP对cas3基因的调控作用。为进一步确认CRP对cas3的功能调控作用，构建Δcrp突变株，通过生物被膜实验、黏附侵袭实验、大蜡螟毒力实验以及小鼠细菌感染模型分析crp对鲍曼不动杆菌毒力的影响。结果 EMSA实验表明CRP蛋白可与cas3基因启动子特异性结合；qPCR结果表明，Δcrp突变株cas3转录水平显著下降(P<0.001)。与野生株相比，Δcrp突变株在生长能力方面无明显差异，但生物被膜形成能力显著增强(P<0.001)，对A549细胞的黏附(P=0.003<0.050)和侵袭(P<0.001)能力也明显增强。Δcrp突变株在72 h内对大蜡螟的致死率显著提高，且小鼠感染实验结果表明，Δcrp突变株在肺部的定殖能力显著高于野生株(P<0.001)。结论 CRP作为一种转录激活因子，可直接结合cas3启动子并激活其转录表达，进而降低鲍曼不动杆菌的毒力和致病性。

Abstract:

Objective To investigate the regulatory effect of the cAMP receptor protein (CRP), a transcription factor in Acinetobacter baumannii, on the cas3 gene.Methods CRP was expressed and purified via a prokaryotic expression system. EMSA was employed to examine CRP binding to the cas3 promoter. qPCR was conducted to evaluate the regulatory effect of CRP on cas3 expression. To further confirm the regulatory function of CRP, we constructed a mutant strain Δcrp. The impact of crp deletion on A. baumannii virulence was then analyzed via the biofilm formation assay, adhesion and invasion assays with A549 cells, a Galleria mellonella model, and a murine model of bacterial infection.Results EMSA demonstrated that CRP specifically bound to the cas3 promoter. The qPCR results showed that cas3 transcription was downregulated (P<0.001) in Δcrp. Compared with the wild-type strain, Δcrp exhibited no significant difference in growth capacity but enhanced biofilm formation (P<0.001) as well as strengthened adhesion (P=0.003<0.050) and invasion (P<0.001) in A549 cells. Furthermore, Δcrp demonstrated a markedly increased lethality rate in G. mellonella within 72 h. Furthermore, the murine infection experiment revealed that Δcrp possessed higher colonization capacity in the lungs than the wild-type strain (P<0.001).Conclusion CRP acts as a transcriptional activator that directly binds to the cas3 promoter to activate its transcription, thereby attenuating the virulence and pathogenicity of A. baumannii.